Pathogenesis, Host Innate Immune Response, and Aerosol Transmission of Influenza D Virus in Cattle

Abstract

The recently discovered influenza D virus (IDV) of the Orthomyxoviridae family has been detected in swine and ruminants with a worldwide distribution. Cattle are considered to be the primary host and reservoir, and previous studies suggested a tropism of IDV for the upper respiratory tract and a putative role in the bovine respiratory disease complex. This study aimed to characterize the pathogenicity of IDV in naive calves as well as the ability of this virus to transmit by air. Eight naive calves were infected by aerosol with a recent French isolate, D/bovine/France/5920/2014. Results show that IDV replicates not only in the upper respiratory tract but also in the lower respiratory tract (LRT), inducing moderate bronchopneumonia with restricted lesions of interstitial pneumonia. Inoculation was followed by IDV-specific IgG1 production as early as 10 days postchallenge and likely both Th1 and Th2 responses. Study of the innate immune response in the LRT of IDV-infected calves indicated the overexpression of pathogen recognition receptors and of chemokines CCL2, CCL3, and CCL4, but without overexpression of genes involved in the type I interferon pathway. Finally, virological examination of three aerosol-sentinel animals, housed 3 m apart from inoculated calves (and thus subject to infection by aerosol transmission), and IDV detection in air samples collected in different areas showed that IDV can be airborne transmitted and infect naive contact calves on short distances. This study suggests that IDV is a respiratory virus with moderate pathogenicity and probably a high level of transmission. It consequently can be considered predisposing to or a cofactor of respiratory disease.IMPORTANCE Influenza D virus (IDV), a new genus of the Orthomyxoviridae family, has a broad geographical distribution and can infect several animal species. Cattle are so far considered the primary host for IDV, but the pathogenicity and the prevalence of this virus are still unclear. We demonstrated that under experimental conditions (in a controlled environment and in the absence of coinfecting pathogens), IDV is able to cause mild to moderate disease and targets both the upper and lower respiratory tracts. The virus can transmit by direct as well as aerosol contacts. While this study evidenced overexpression of pathogen recognition receptors and chemokines in the lower respiratory tract, IDV-specific IgG1 production as early as 10 days postchallenge, and likely both Th1 and Th2 responses, further studies are warranted to better understand the immune responses triggered by IDV and its role as part of the bovine respiratory disease complex.

Keywords: BRD; cattle; influenza virus D; pathogenicity; respiratory; transmission.

FIG 1

(A) Experiment timeline and sampling. D0 is the day of inoculation. (B) Fourteen naive calves were allocated to two separated pens, one with 5 mock-infected calves (control group) and the second with 11 calves. In the latter pen, calves were divided into two groups separated by steel panels, 3 m apart. Eight calves (direct-inoculated group) were inoculated at day 0 (D0) with 10⁷ TCID₅₀ of IDV 5920, while the three others (aerosol-sentinel group) were not, in order to evaluate the aerosol transmission of IDV.

FIG 2

Mean clinical scores (A) and mean accumulated clinical scores (ACS, mean of the individual area under daily clinical scores using the trapezoid method) (GraphPad, La Jolla, CA) (B) of the five IDV direct-inoculated animals that were kept beyond day 8, all control uninfected calves, and all aerosol-sentinel calves.

FIG 3

(A) Subacute bronchointerstitial pneumonia with neutrophils in bronchial lumens, neutrophilic and macrophagic alveolitis and peribronchial and septal lymphoplasmocytic infiltration (left; magnification, ×200) and microscopic alveolar lesions at a higher magnification (right; magnification, ×400). (B) (Left) Immunostaining of left cranial lobe of calf no. 7165 inoculated with IDV showing cytoplasmic and nuclear labeling of the bronchiolar epithelium by the anti-IDV polyclonal antibody. (Right) Negative control of the same slide with the substitution of the IDV polyclonal serum by a rabbit normal serum (magnification, ×200).

FIG 4

RT-qPCR IDV RNA loads in nasal secretions (A) and BALs (B) in the five IDV direct-inoculated animals that were kept beyond day 8 (blue), control animals (orange), and aerosol-sentinel calves (green). (C) IDV RNA loads were also detected in air samples collected over the course of the experiment in the direct-inoculated group area (infected area), the 3-m space between direct-inoculated and aerosol-sentinel calves (separated area), and the aerosol-sentinel area (contact area). Dashes represent negative results.

FIG 5

Antibody response in the five IDV direct-inoculated animals that were kept beyond D8 (blue) or all control (orange) calves assessed by IHA (A) and by specific ELISAs for IDV IgG1 (blue) or IgG2 (green) (B). The line in panel B represents the positive threshold of ELISAs.

FIG 6

IDV-specific lymphocyte proliferative responses of PBMC from all directly inoculated calves that were kept beyond D8. The cells were collected at D−1 and D22 and stimulated ex vivo with either heat-inactivated influenza D virus-infected or uninfected cell lysate (A) or untreated influenza D virus-infected or uninfected cell lysate (B). After 6 days of incubation, proliferative response was determined by corrected optical density (COD) of alamarBlue (Invitrogen, Sweden). Results are expressed as the mean COD of triplicate samples, with standard deviations indicated by upward deflecting lines.